LOCUS       sequence-328896-        7300 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for generation of N-terminal His10-Smt3 
            and C-terminal SNAP tagged proteins in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7300)
  AUTHORS   Harel I, Chen YR, Ziv I, Singh PP, Heinzer D, Navarro Negredo P, 
            Goshtchevsky U, Wang W, Astre G, Moses E, McKay A, Machado BE, 
            Hebestreit K, Yin S, Sanchez Alvarado A, Jarosz DF, Brunet A
  TITLE     Identification of protein aggregates in the aging vertebrate brain 
            with prion-like and phase-separation properties.
  JOURNAL   Cell Rep. 2024 Jun 25;43(6):112787. doi: 
            10.1016/j.celrep.2023.112787. Epub 2024 May 28.
  PUBMED    38810650
REFERENCE   2  (bases 1 to 7300)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Rep.";
            date: "2024-06-25"; volume: "43(6):112787. doi"; pages: " 
            10.1016/j.celrep.2023.112787. Epub 2024 May 28"
COMMENT     Sequence Label: pDEST17-His10-Smt3-ccdB-SNAP
FEATURES             Location/Qualifiers
     source          1..7300
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             71..93
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             113..142
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             155..166
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             179..472
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     protein_bind    485..609
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(634..639,640..657,658..664)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             718..1377
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(784..803)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1719..2024
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1903..1922
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2065..2189)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2191..2214
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2233..2778
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for bacterial expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     primer_bind     complement(2846..2864)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2860..2907
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3273..3278,3279..3295,3296..3301))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3328..3346)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3414..3518
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3519..3587,3588..4379)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3737..3756)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4550..5138
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5039..5058
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5292..5309
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5324..5463
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5389..5408)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5549..5571
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5565..5756)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     7218..7237
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agatctcgat cccgcgaaat taatacgact cactataggg agaccacaac ggtttccctc
       61 tagaaataat tttgtttaac tttaagaagg agatatacat atgtcgtact accatcacca
      121 tcaccatcac catcatcatc acagcagcgg ccatatcgaa ggtcgtcata tggctagcat
      181 gtcggactca gaagtcaatc aagaagctaa gccagaggtc aagccagaag tcaagcctga
      241 gactcacatc aatttaaagg tgtccgatgg atcttcagag atcttcttca agatcaaaaa
      301 gaccactcct ttaagaaggc tgatggaagc gttcgctaaa agacagggta aggaaatgga
      361 ctccttaaga ttcttgtacg acggtattag aattcaagct gatcagaccc ctgaagattt
      421 ggacatggag gataacgata ttattgaggc tcacagagaa cagattggtg gatccctcga
      481 atcaacaagt ttgtacaaaa aagctgaacg agaaacgtaa aatgatataa atatcaatat
      541 attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac aacatatcca
      601 gtcactatgg cggccgcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
      661 aatgtgtgga ttttgagtta ggatccgtcg agattttcag gagctaagga agctaaaatg
      721 gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat
      781 tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt
      841 acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac
      901 attcttgccc gcctgatgaa tgctcatccg gaattccgta tggcaatgaa agacggtgag
      961 ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca aactgaaacg
     1021 ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca catatattcg
     1081 caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt tattgagaat
     1141 atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt aaacgtggcc
     1201 aatatggaca acttcttcgc ccccgttttc accatgggca aatattatac gcaaggcgac
     1261 aaggtgctga tgccgctggc gattcaggtt catcatgccg tttgtgatgg cttccatgtc
     1321 ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg ggcgtaaaga
     1381 tctggatccg gcttactaaa agccagataa cagtatgcgt atttgcgcgc tgatttttgc
     1441 ggtataagaa tatatactga tatgtatacc cgaagtatgt caaaaagagg tatgctatga
     1501 agcagcgtat tacagtgaca gttgacagcg acagctatca gttgctcaag gcatatatga
     1561 tgtcaatatc tccggtctgg taagcacaac catgcagaat gaagcccgtc gtctgcgtgc
     1621 cgaacgctgg aaagcggaaa atcaggaagg gatggctgag gtcgcccggt ttattgaaat
     1681 gaacggctct tttgctgacg agaacagggg ctggtgaaat gcagtttaag gtttacacct
     1741 ataaaagaga gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc
     1801 ccgggcgacg gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc
     1861 gtgaacttta cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata
     1921 tggccagtgt gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa
     1981 atgacatcaa aaacgccatt aacctgatgt tctggggaat ataaatgtca ggctccctta
     2041 tacacagcca gtctgcaggt cgaccatagt gactggatat gttgtgtttt acagtattat
     2101 gtagtctgtt ttttatgcaa aatctaattt aatatattga tatttatatc attttacgtt
     2161 tctcgttcag ctttcttgta caaagtggtt gactacaagg atgacgatga caagcttacc
     2221 ggtgagttcg ctatggacaa agattgcgaa atgaaacgta ccaccctgga tagcccgctg
     2281 ggcaaactgg aactgagcgg ctgcgaacag ggcctgcatg aaattaaact gctgggtaaa
     2341 ggcaccagcg cggccgatgc ggttgaagtt ccggccccgg ccgccgtgct gggtggtccg
     2401 gaaccgctga tgcaggcgac cgcgtggctg aacgcgtatt ttcatcagcc ggaagcgatt
     2461 gaagaatttc cggttccggc gctgcatcat ccggtgtttc agcaggagag ctttacccgt
     2521 caggtgctgt ggaaactgct gaaagtggtt aaatttggcg aagtgattag ctatcagcag
     2581 ctggcggccc tggcgggtaa tccggcggcc accgccgccg ttaaaaccgc gctgagcggt
     2641 aacccggtgc cgattctgat tccgtgccat cgtgtggtta gctctagcgg tgcggttggc
     2701 ggttatgaag gtggtctggc ggtgaaagag tggctgctgg cccatgaagg tcatcgtctg
     2761 ggtaaaccgg gtctgggacc tgcaggctga taaggattcg aggctgctaa caaagcccga
     2821 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     2881 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg atatccacag
     2941 gacgggtgtg gtcgccatga tcgcgtagtc gatagtggct ccaagtagcg aagcgagcag
     3001 gactgggcgg cggccaaagc ggtcggacag tgctccgaga acgggtgcgc atagaaattg
     3061 catcaacgca tatagcgcta gcagcacgcc atagtgactg gcgatgctgt cggaatggac
     3121 gatatcccgc aagaggcccg gcagtaccgg cataaccaag cctatgccta cagcatccag
     3181 ggtgacggtg ccgaggatga cgatgagcgc attgttagat ttcatacacg gtgcctgact
     3241 gcgttagcaa tttaactgtg ataaactacc gcattaaagc ttatcgatga taagctgtca
     3301 aacatgagaa ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     3361 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     3421 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3481 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     3541 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     3601 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     3661 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     3721 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc
     3781 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     3841 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     3901 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     3961 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     4021 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt
     4081 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4141 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4201 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     4261 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4321 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4381 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4441 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4501 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4561 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4621 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4681 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4741 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     4801 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4861 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4921 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4981 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5041 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5101 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5161 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5221 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     5281 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     5341 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     5401 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     5461 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     5521 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     5581 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     5641 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     5701 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     5761 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     5821 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     5881 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     5941 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     6001 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     6061 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     6121 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     6181 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg
     6241 cccgagatgc gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa
     6301 gggttggttt gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg
     6361 gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg
     6421 caccgcgacg caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca
     6481 acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccaatga
     6541 tcgaagttag gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat
     6601 ctacctgcct ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa
     6661 gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca
     6721 gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     6781 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     6841 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     6901 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     6961 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgat
     7021 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     7081 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     7141 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     7201 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     7261 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
//
